Detection of red tide organisms by nucleic acid amplification

ABSTRACT

A real-time reverse transcription-PCR, or NABSA, method targeting the rbcL gene developed for the detection and quantitation of the Florida red tide organism,  Karenia brevis.

CROSS-REFERENCE TO RELATED DISCLOSURES

This application claims priority of a provisional application of thesame title, filed Jan. 8, 2003 by the present inventors and bearingapplication No. 60/319,841.

STATEMENT OF GOVERNMENT INTEREST

The work that led to this invention has been supported in part by agrant from the National Oceanic & Atmospheric Administration, GrantNumber NA160p1437. Thus, the United States Government may have certainrights to this invention.

BACKGROUND OF THE INVENTION

Karenia brevis is an unarmored, non-peridinin-containing dinoflagellatethat grows to ca. 20 to 40 μm in diameter. The organism is positivelyphototactic, is negatively geotactic, swims at a speed of ca. 1 m h⁻¹and is thought to be an obligate photoautotroph. K. brevis is thecausative agent of the recurring red tide blooms (21 of 22 years from1975 to 1997) observed in the Gulf of Mexico and off the southeasternAtlantic coast of the United States, which have been reported since theSpanish conquests. Lipophilic brevetoxins produced by K. brevis canresult in massive fish kills and have been implicated in the mortalityof 700 bottlenose dolphin off the east coast of the United States in1987 and the mysterious deaths of 149 Florida manatees in 1995 and 1996.In cases of human exposure, brevetoxin can cause respiratory distress byinhalation and food poisoning by consumption of tainted shellfish.

Current methods for the detection of K. brevis depend on microscopy orpigment analysis, methods which are time-consuming and require aconsiderable amount of expertise and skill. Isolation of dinoflagellatesand cultivation from environmental samples to confirm identity may takemonths. Consequently, rapid molecular methods to detect K. brevis in theenvironment are needed.

SUMMARY OF INVENTION

The present invention is a method of detecting the presence of at leastone dinoflagellate, specifically K. brevis, in a water sample comprisingthe steps of, identifying a unique gene sequence associated with theorganism K. brevis, amplifying the unique gene sequence contained in thewater sample using at least one purified primer, the primer having atleast one K. brevis specific internal probe affixed thereto, theinternal probe having a label attached thereto, and detecting thepresence of the unique gene sequence within the sample.

Although many embodiments of the invention are contemplated, in oneembodiment the amplification step is performed by reverse transcriptasepolymerase chain reaction. When using reverse transcriptase polymerasechain reaction the unique gene sequence is a 91-base-pair region of theK. brevis rbcL gene. The purified primer sequence for real time reversetranscriptase polymerase chain reaction is SEQ ID NO: 1 (forward primer)and SEQ ID NO: 2 (reverse primer). The internal probe for real timereverse transcriptase polymerase chain reaction is SEQ ID NO: 3.Finally, the label attached to the internal probe is a flourogeniccompound and detection is achieved utilizing epiflouresence microscopy.

In another embodiment, the amplification step is performed by nucleicacid based amplification (NASBA). When using the NASBA method the uniquegene sequence is an 87-base-pair region of the K. brevis rbcL gene. Thepurified primer sequence for nucleic acid sequence based amplificationis SEQ ID NO: 4 (P1) and SEQ ID NO: 5 (P2). Finally, the internal probe(Beacon) for nucleic acid sequence based amplification is SEQ ID NO: 6.

BRIEF DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and objects of the invention,reference should be made to the following detailed description, taken inconnection with the accompanying drawings, in which:

FIG. 1. indicates the positive and negative controls for amplificationby real time RT_PCR.

FIG. 2. shows a neighbor-joining phylogenetic tree based on deducedamino acid sequences with a Poisson distance correction showingrelationships between form I rbcL sequences from K. brevis and otherphytoplankton species, as well as clones obtained on a cruise to theMississippi River plume in the Gulf of Mexico. Boldface taxa were testedby real-time RT-PCR as nontarget controls. There were many taxa testedas nontarget strains whose rbcL sequences were not available in GenBank,and closest sequenced representatives are underlined.

FIG. 3. depicts a real-time RT-PCR standard curve generated from theAPC6 clone 15 transcript showing the linearity of the method, covering 7orders of magnitude (filled circles [trendline]). Also shown aream-plification results from K. brevis cellular extracts corresponding to100 cells, 10 cells, and 1 cell per reaction (open circles).

FIG. 4 shows a comparison of microscopy cell counts and real-timeRT-PCR-inferred cell counts from natural bloom samples.

FIG. 5 is a graphic representation of the nucleic acid sequence basedamplification (NASBA) pathway.

FIG. 6 is an illustration of a molecular beacon. Unbound the quencher(3′) prevents the dye (5′) from fluorescing. Once bound to the amplicon,the dye and quencher are separated and the dye gives off a fluorescentsignal.

FIG. 7 represents the real time amplification plot and standard curve ofK. brevis. Standard curve was generated from 10³, 10², 10, and 1 fgtranscript RNA. Unknowns are a combination of cultured cells and bloomsamples containing varying numbers of K. brevis.

FIG. 8 is an EM photograph of K. brevis.

FIG. 9 is a graphic representation of the inventive method.

BRIEF DESCRIPTION OF SEQUENCES

<110> University of South Florida

<120> DETECTION OF RED TIDE ORGANISMS BY NUCLEIC ACID AMPLIFICATION

<130> 1372.120PCR

<160> 8

<170> Patent In version 3.2

<210> 1

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Forward primer designed to amplify and detect the 91-bp region ofthe rbcL gene specific to K. brevis.

<400> 1

tgaaacgtta ttgggtctgt 20

<210> 2

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Reverse primer designed to amplify and detect the 91-bp region ofthe rbcl gene specific to K. brevis.

<400> 2

aggtacacac tttcgtaaac ta 22

<210> 3

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Fluorogenic probe designed to amplify and detect the 91-bp regionof the rbcl gene specific to K. brevis.

<400> 3

ttaaccttag tctcgggta 19

<210> 4

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Real Time NASBA primer (1) for the marker region of rbcL genespecific to K. brevis.

<400> 4

acgttattgg gtctgtgta 19

<210> 5

<211> 50

<212> DNA

<213> artificial sequence

<220>

<223> Primer (2) for real time NASBA to detect the marker region of thercbL gene specific to K. brevis.

<400> 5

aattctaata cgactcacta tagggagaag gtacacactt tcgtaaacta 50

<210> 6

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> Molecular beacon used for real time NASBA assay.

<400> 6

cgatcgctta gtctcgggtt attttttcga tcg 33

<210> 7

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> PCR primer set was designed with sequence data from Kareniamikimotoi (GenBank accession no. ABO34635) by modifying existingchromophyte rbcL primers in order to amplify a 554-bp region(approximately one-third) of Karenia's rbcL gene.

<400> 7

atgatgaraa yattaactc 19

<210> 8

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> PCR primer set was designed with sequence data from Kareniamikimotoi (GenBank accession no. ABO34635) by modifying existingchromophyte rbcL primers in order to amplify a 554-bp region(approximately one-third) of Karenia's rbcL gene.

<400> 8

atttgtcccg cattgattcc t 21

DETAILED DESCRIPTION

A molecular marker for this organism is ribulose-1,5-bisphosphatecarboxylase/oxygenase (RuBisCO) large-subunit gene (rbcL). RuBisCO isthe primary carbon-fixing enzyme in photoautotrophic organisms. K.brevis and the other fucoxanthin-containing dinoflagellates have a formID rbcL enzyme, and genetic evidence suggests that they contain plastidsof haptophyte origin acquired through tertiary endosymbiosis.

As rbcL is highly expressed in viable cells and mRNA levels can beorders of magnitude greater than those of DNA, the mRNA was targeted forthis study. As RNA is rapidly degraded in the environment, an RNA targetwill give an indication of a viable population compared to what isdetected by DNA-based methods, which may detect dead cells as well.

PCR Method

To obtain sequence data for this embodiment of the inventive method, aPCR primer set was designed with sequence data from Karenia mikimotoi(GenBank accesno. ABO34635) by modifying existing chromophyte rbcLprimers in order to amplify a 554-bp region (approxone-third) ofKarenia's rbcL gene (forward primer, ATGATGARAAYATTAACTC, (SEQ. ID NO.7)); reverse primer, ATTTGT CCCGCATTGATTCCT, (SEQ. ID NO. 8),[International Union of Pure and Applied Chemistry degeneracy symbolswere used]).

Cultures of K. brevis were obtained from the Florida Fish and WildlifeConservation Commission's Florida Marine Research Institute. Strainswere isolated by Marine Research Institute lab from the followinglocations around the Florida coast: Apalachicola, Charlotte Harbor,Mexico Beach, Jackand Piney Island. Strains used in this analysis werenamed for their isolation location and the plate well into which theywere isolated. Several nontarget algal strains of diverse lineage wereobtained from either the Provasoli-Guillard Cenfor Culture of MarinePhytoplankton (CCMP; West Booth-bay Harbor, Me.) or from the Steidingerlab (FIG. 1). All strains were under a 12-h-light12-h-dark light regimenat 26 μmol s⁻¹ m² and were incubated at 20 or 14° C. in F/2 medium,which was modified for each strain's needs acto CCMP's directions.

K. brevis cells are harvested by centrifugation (10 min at 5,000×g), andthe DNA extracted by a modified phenol-chloroform method. PCRamplification is conducted with final concentrations of 1 μM for theprimers, 3 mM for MgCl2, 0.4 mM for each deoxynucleoside triphosphate,and 2.5 U of Taq polymerase (Promega Corp., Madison, Wis.). Cyclingconditions are 40 repetitions of 95° C. for 1 min, 50° C. for 1 min, and72° C. for 1.5 min, with a final extension step at 72° C. for 15 min.Amplification is confirmed by agarose gel electrophoresis PCR ampliconsare purified with a QIAquick PCR purikit (QIAGEN, Valencia, Calif.) andligated into the pCR II vector, and TOP10 cells transformed according tothe manufacturer's instructions (Invitrogen Corp., Carlsbad, Calif.).Transformants are plated onto 2XYT plates containing 50 μg (each) ofkanamycin and ampicillin per ml. White colonies are screened for insertsize by PCR amplification. Positive clones are grown in 2XYT broth withantibiotics, and plasmid DNA extracted with a Wizard Plus SV miniprepspin kit (Promega Corp.). Clones from nontarget species from a rbcLclone library are also grown and extracted as described above.Sequencing of the 554-bp K. brevis and K. mikimotoi insert are performedat the DNA Sequencing Core laboratory at the University of Florida.

One of the sequenced clones carrying the 554-bp insert from K. brevisAPC6 (clone 15) can be used in sensitivity testing. Nontargetenvironmental rbcL clones (from the same region of the gene) wereobtained from the Gulf of Mexico during a previous study to initiallytest specificity (FIG. 1). Based on the direction of the insert, thevector is linearized by digesting the plasmid with either HindIII orEcoRV and a sense transcript made by in vitro transcription using the T7or SP6 promoter site. The transcripts are purified with a QIAGEN RNeasyRNA extraction kit, with the DNase digestion step being performedaccording to the manufacturer's instructions. These transcripts arequantified with a Ribogreen RNA quantification kit according to themanufacturer's instructions (Molecular Probes, Inc., Eugene, Oreg.),mixed 1:1 with an RNA storage buffer (8 M guanidinium isothiocyanate 80mM Tris-HCl [pH 8.5], 24 mM MgCl2, 140 mM KCl), aliquoted, and frozen at−80° C. The K. brevis APC6 clone 15 transcript is used to generatereal-time reverse transcription (RT)-PCR standard curves, while theothers are used to test the specificity of the primer-probe set.

Sequences for phylogenetic comparison can be obtained from GenBank.During the course of experimentation, a sequence for K. brevis appearedin GenBank. Sequences were aligned and analyzed using the KODON softwarepackage, version 1.0 (Applied Maths, Inc., Austin, Tex.), which uses aClustal W alignment method. Phylogenetic and molecular evolutionaryanalysis is conducted using MEGA2 software (version 2.1; S. Kumar, K.Tamura, I. B. Jakobsen, and M. Nei, Arizona State University, Tempe,2001) using both nucleotide and deduced amino acid sequence data. Allnontarget strains, their representative accession numbers, and theirrelationships based on deduced amino acid residues are shown in FIG. 2.

Sequence data from the K. brevis rbcL clones show a short (91-bp) regionthat is markedly different from K. mikimotoi's rbcL sequence. Thisportion of the rbcL gene of K. brevis has been selected as the targetfor a primer and probe set for the Taq-Man Taq nuclease assay. A primerset and an internal fluoroprobe were designed to amplify and detect the91-bp region (forward primer, TGAAACGT-TATTGGGTCTGT, (SEQ. ID NO. 1);reverse primer, AGGTA-CACACTTTCGTAAACTA, (SEQ. ID NO. 2); internalprobe, FAM [6-carboxyfluorescein]-TTAACCTTAGTCTCG GGTA-TAMRA, (SEQ. IDNO. 3), [6-carboxytetramethylrhodamine]). For real-time RT-PCR, 5 μl ofthe target is added to 45 μl of a one-step RT-PCR mixture prepared from2×RT-PCR TaqMan master mix (Applied Biosystems, Foster City, Calif.)containing each primer at a concentration of 1 μM, 2 mM MgCl, and a 0.5μM concentration of the probe. Cycling conditions are as follows: aprecycling reverse transcription step of 45° C. for 30 min; an initialdenaturation step of 95° C. for 10 min; and then 40 cycles of 95° C. for1 min, 55° C. for 1 min, and 72° C. for 1 min. Reaction mixtures run inthe Applied Biosystems 7700 sequence detection system and are analyzedwith their supplied software.

Cell counts for all cultured algal strains (including K. brevis) arecarried out by filtering 1 ml of culture onto 0.22-μm-pore-size blackpolycarbonate Poretics filters (Osmonics Inc., Minnetonka, Minn.). Cellsare counted by using epifluorescence microscopy on an Olympus BX-60microscope with the 20Xobjective and blue excitation (filter setU-MNIB). RNA from the culture is extracted using the QIAGENRNeasy spinkit with the following modifications. Culture samples (1 ml) arefiltered onto a 0.45-μm-pore-size HV polyvinylidene difluoride filter(Millipore Durapore). The filters are placed into 2-ml screw-capmicrocentrifuge tubes containing 750 μl of RLT lysis buffer (QIAGEN)with 2-mercap-toethanol (10 μlml⁻¹). The filters are then incubated for10 min at room temperature, 500 μl is removed into a 1.5-ml mitube, andRNA extraction continued according to the manufacturer's instructions(QIAGEN). The extracted RNA can be quantified using a Ribogreen RNAquantification kit according to the manufacturer's instructions. Allnontarget algal strains are tested for amplification with the real-timeprimer-probe set with 10 pg of nontarget RNA per reaction mixture.

Field samples were collected by the Florida Marine Research Institutefrom several locations at several different times in Collier County(west coast of Florida; collected 28 Mar., 2 and 9 Apr., and 2 May 2003)and from the Indian River lagoon (east coast of Florida; collected 13Dec. 2002) during both bloom and nonbloom events. Algae in field sampleswere counted by microscopy by the Florida Fish and Wildlife ConservationCommission prior to our receiving them. Algae from field samples wereextracted as described above, but 10 to 20 ml was extracted and 5 μl ofthe extract was added to the RT-PCR mixture.

The TaqMan probe-based RT-PCR assay (91-bp amplicon) yields onlypositive results with K. brevis strains (FIG. 2). All otherdinoflagellates (including K. mikimotoi) and algal strains resulted inno amplification. All strains tested were present in sufficientconcentrations to allow for amplification based on the lowest detectableconcentration of K. brevis. Standard curves derived by using the invitro transcript from APC6 clone 15 show sensitivity over a range ofconcentrations spanning 7 orders of magnitude, ranging from 0.1 fg to1,000 pg, as shown in FIG. 3. Standard curves using whole-cell extractsfrom K. brevis culture are sensitive to as little as 1 pg of total RNA(less than 1 cell per reaction, based on cell counts and dilution).

Red tide bloom and nonbloom samples from around Florida were analyzedfor K. brevis using this method. From the west coast, 15 samples wereanalyzed; 11 were non-bloom and 4 were moderate to high bloom. The twosamples from the east coast were composed of one bloom and one nonbloom.Microscopy counts of the nonbloom samples were below the detection limitof 333 cells liter⁻¹. Counts inferred by RT-PCR were mostly 0.0 cellliter⁻¹ or below the detection limit by microscopy (7 of 12 samples). Ofthe remaining five nonbloom samples, three gave a result ofapproximately 1,000 cells liter⁻¹, one indicated 3,000 cells liter, andone indicated 12,000 cells liter⁻¹. The last sample's result may be dueto contamination of the sample. For the bloom samples, all but oneindicated that cell density was very close to that of the microscopycounts, and one sample indicated approximately one-third the density bymicroscopy. As this method targets mRNA, it is possible that the cellsin the last sample were not producing high levels of transcript or thatthey were no longer viable. FIG. 4 summarizes this comparison of celldensities for these field samples as enumerated by microscopy andinferred from real-time RT-PCR. A good correlation (r²=0.878) wasobserved between the results of both methods for field samples.

Using the TaqMan probe, it is possible to amplify and detect a widerange of concentrations of K. brevis to the exclusion of all nontargetDNA and RNA tested, with a detection limit of less than 100 cellsliter⁻¹ when 20 ml of seawater is extracted. When larger volumes arefiltered, lower detection limits should be attainable. The dynamic rangeover which this technique is effective covers the range of natural K.brevis blooms in the environment. When an environment contains <1,000cells liter⁻¹ (as determined by microscopy cell counts), K. brevis isconsidered to be present but poses no risk of adverse health effects orshellfish contamination. Samples with >1,000 cells liter⁻¹ areconsidered to have a very low level bloom, carrying a slight risk ofrespiratory irritation. At concentrations of >5,000 cells liter⁻¹shellfish harvesting is closed. The highest level of a bloom has beenreached when there are >10⁶ cells liter⁻¹. A bloom of this magnitude canresult in massive fish kills, respiratory distress in humans, anddiscoloration of the water and can affect the health of marine mammalssuch as dolphins and manatees.

NASBA Method

Nucleic acid sequence-based amplification (NASBA) (FIG. 5), is anisothermal method of RNA amplification (Davey and Malek, 1989). At afixed temperature of 41° C., RNA is amplified by use of an enzymecocktail consisting of T7 RNA polymerase, AMV reverse transcriptase, andRnaseH and two target specific oligonecleotide primers. Primer P1 (SEQ.ID NO. 4), which contains T7 RNA polymerase promoter hybridizes to thetarget RNA and a cDNA is made by the AMV reverse transcriptase. Thisresults in the production of a DNA/RNA hybrid. RnaseH digests the RNA,leaving a single strand of antisense DNA. Primer 2 (SEQ. ID NO. 5) thenbinds to the DNA and is elongated, yielding a double stranded DNAmolecule with an intact T7 RNA polymerase promoter sequence. From thisdouble stranded product, many copies of single stranded anti-sense RNAare transcribed. This antisense RNA serves as the template for a newcycle of amplification, though the primers bind in reverse order.

In this embodiment of the inventive method, real time detection of theamplicon is accomplished by use of a molecular beacon (SEQ. ID NO. 6)labeled with 6-carboxy fluorescein (6-FAM) at its 5′ end and quencherDABCYL at its 3′ end (FIG. 6). The beacon forms a stem-loop structure.The loop sequence is complementary to the target sequence and twocomplementary stem sequences are added to each side of the loop. Whenbound to amplicon, the quencher is separated from the fluorescein, andthe probe fluoresces. The fluorescent signal is measured and the time atwhich it reaches a threshold of detection is measured. The time it takesa sample to reach the threshold is a function of how much initial targetRNA is in the sample.

NASBA primers for detection of K. brevis are designed to target an 87base pair sequence of the ribulose 1,5-bisphosphatecarboxylase-oxygenase large subunit (rbcL). Standard curves andsensitivity are determined with in vitro transcript as well as withwhole cells from culture. Specificity is assessed using non-targetenvironmental rbcL clones and environmental isolates. Sequenceinformation for rbcL of clones and isolates are obtained from GenBankand nucleotide sequences are aligned using the Clustal W algorithm(Thompson et al., 1994) in Kodon v1.0 (Applied Maths Inc, Austin Tex.).Amino acid sequences deduced from the nucleotide sequences were comparedand a phylogenetic tree was created by performing neighbor joining with1000 bootstrap replications in the MEGA v2.1 (Kumar et al., 1993)software package.

Cells from culture and natural seawater samples are filtered ontosterile Millipore Durapore 0.45 μm filters prior to RNA extraction viathe RNeasy Kit from Qiagen. The filters are submerged in the Qiagensupplied RLT lysis buffer for 10 minutes. After incubation the buffer isrecovered and the RNeasy protocol followed.

Karenia brevis cultures and natural seawater samples were obtained fromthe staff at the Florida Marine Research Institute (FMRI). Naturalseawater samples were collected by a variety of agencies in SouthwestFlorida as part of a regular monitoring program. Non-target organismswere obtained from B. Richardson (FMRI) and the Provasoli-GuillardNational Center for Culture of Marine Phytoplankton (CCMP).Environmental clones were supplied by B. Wawrik (University of SouthFlorida).

As a result of the inventive method:

Amplification of cultured cells occurred in about 15 minutes (FIG. 7).

The assay was sensitive to as little as one K. brevis cell and to datecan detect 1 fg in vitro transcript.

Karenia brevis was detected in natural seawater samples collected whereblooms had been reported.

The NASBA assay did not result in detection of non-target organisms andenvironmental clones.

This embodiment of the inventive method is a real time NASBA assays forthe detection and quantification of marine enteroviruses and K. brevis.This assay can routinely detect 100 viral particles and can detect aslittle as one K. brevis cell. NASBA is a rapid and sensitive method fordetection of RNA, and can be applied to a variety of organisms. NASBA isused in clinical settings to detect a variety of microorganisms,including HIV, hepatitis, Salmonella, and E. coli, among many others.NASBA has also been used to detect other microorganisms in aquaticenvironments. This embodiment of the inventive method is the first toapply NASBA to the detection of K. brevis from marine waters.

NASBA is both rapid and sensitive and is less technologically demandingthan other molecular detection methods. Advantages of using NASBAinclude its isothermal nature and rapid, real time detection. Rapiddetection of these microorganisms is important from both human healthand economic standpoints. These assays are being developed with theeventual goal of being used in the field (at sea or in coastal waters)with the assistance of personnel or being deployed on an autonomoussensor platform (moored buoy or autonomous underwater vehicle). Thistechnology is important as it will enable the rapid detection,identification, and quantification of microorganisms in coastal waters.

For Real Time NASBA, the following primers and molecular beacon areused:

Breve NASBA1-5′-ACGTTATTGGGTCTGTGTA-3′

BreveNASBA2-5′-AATTCTAATACGACTCACTATAGG GAGA-AGGTACACAC TTTCGTAAACTA-3′

BreveBEACON-5′-FAM-CGATCGCTTAGTCTCGGGTTATTTTTTCGATCG-DA BCYL-3′

FIG. 9 is a graphic representation of the inventive method. First theunique gene sequence is identified 10. Then a water sample is taken 20.The target gene sequence is amplified, by either polymerase chainreaction or nucleic acid sequence based amplification, using thespecific primers and probes 30. After the amplification process iscomplete the sample is tested for the presence of the amplified sequence40.

These methods represent the first molecular detection strateg for K.brevis, and are well suited for the detection and monitoring of red tideblooms caused by K. brevis in the Gulf of Mexico and the southernAtlantic coast of the United States. Although diel regulation of rbcL inK. brevis has not been characterized, this assay provides an easy andrelatively rapid procedure that might be employed as an alternative tothe more difficult and time-consuming methods currently used by red tidemonitoring and management programs in Florida and other states affectedby K. brevis. It will be seen that the objects set forth above, andthose made apparent from the foregoing description, are efficientlyattained and since certain changes may be made in the above constructionwithout departing from the scope of the invention, it is intended thatall matters contained in the foregoing description or shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense. It is also to be understood that the following claimsare intended to cover all of the generic and specific features of theinvention herein described, and all statements of the scope of theinvention which, as a matter of language, might be the to falltherebetween. Now that the invention has been described,

1. A method for screening a sample for the presence of K. brevis,comprising: subjecting the sample to amplification using a pair ofoligonucleotide primers capable of amplifying a target region of theribulose 1,5-biphosphate carboxylase-oxygenase large subunit (rbcL) ofK. brevis; and assaying the mRNA for the presence of the amplifiedtarget region of the ribulose 1,5-biphosphate carboxylase-oxygenaselarge subunit (rbcL) unique to K. brevis.
 2. The method of claim 1wherein the pair of oligonucleotide primers specifically amplify mRNA ofa target region of the ribulose 1,5-biphosphate carboxylase-oxygenaselarge subunit (rbcL) of K. brevis and do not amplify a region of theribulose 1,5-biphosphate carboxylase-oxygenase large subunit (rbcL of K.mikimotoi.
 3. The method of claim 1 wherein the target region of theribulose 1,5-biphosphate carboxylase-oxygenase large subunit (rbcL) ofK. brevis is about 87 to 91 base pairs in length.
 4. The method of claim1 wherein the amplification process is selected from the groupconsisting of real-time reverse-transcriptase polymerase chain reactionand quantitative thermocycling.
 5. The method of claim 4 wherein thepair of oligonucleotide primers consist of SEQ. ID. No. 1 and SEQ. ID.No.
 2. 6. The method of claim 5 wherein the pair of oligonucleotideprimers are specific to a target region of the ribulose 1,5-biphosphatecarboxylase-oxygenase large subunit (rbcL) of K. brevis about 91 basepairs in length.
 7. The method of claim 5 wherein the amplificationprocess is applied to the sample in the presence of a probe.
 8. Themethod of claim 7 wherein the probe consists of SEQ. ID. No.
 6. 9. Themethod of claim 1 wherein the amplification process is real time nucleicacid sequence based amplification.
 10. The method of claim 9 wherein thepair of oligonucleotide primers consist of SEQ. ID. No. 4 and SEQ. ID.No.
 5. 11. The method of claim 9 wherein the amplification process isapplied to sample in the presence of a probe.
 12. The method of claim 11wherein the probe comprises a nucleotide sequence consisting of SEQ. ID.No.
 3. 13. The method of claim 9 wherein the pair of oligonucleotideprimers is specific to a target region of the ribulose 1,5-biphosphatecarboxylase-oxygenase large subunit (rbcL) of K. brevis about 87 basepairs in length.